Electrophoretic mobility shift analysis for BmEcRB1-E1 and E2.
(A, B) Competition assay with cold probes for BmEcRB1-E1 (A) and for BmEcRB1-E2 (B). Mutation sites in E1 and E2 probe sequence “N” (Normal) are shown in gray region of “M” (Mutant). Two hundred (A) or 50 (B) femtomoles of 32P-probe were incubated with 5 µg (A) and 10 µg (B) of cell extracts and loaded onto the gel. 20E (6 h) represents extracts from cells cultured under 20E (2.0 µg/mL). ×1, ×10, ×50 and ×100 represent the ratio of the cold probe amount to the 32P-probe amount. Filled arrows show the shifted bands and blank arrows show the free probes. (C, D) Super shift assay with the anti-V5 or/and anti-USP antibody for E1 (C) and for E2 (D). Intact: intact cell extracts. anti-V5 AB: anti-V5 antibody. anti-USP AB: anti-USP antibody. AT, B1T, and USPT represent extracts from cells that overexpressed EcRA, EcRB1, and USP, respectively.